One of the basic tools of molecular biology are proteins with a clearly defined activity, used for example in genetic engineering, diagnostics, medicine and industry in the manufacturing and processing of various products.
DNA restriction endonucleases are sequence dependent enzymes that recognize and cleave specific sequence of double-stranded DNA. There are also known enzymes that cleave RNA in a given sequence, however, such enzymes act on single-stranded sites in RNA. Examples of these enzymes include a phage protein RegB, which cleaves the single-stranded RNA in the middle of the sequence GGAG and Ribonuclease Y, which cleaves the single-stranded RNA in A or AU rich sequences. These enzymes require additional determinants for efficient cleavage, such as RNA secondary structure and in case of RegB the interaction with the ribosomal protein S1 (Lebars, I., et al., J Biol Chem (2001) 276, 13264-13272, Saida, F. et al., (2003) Nucleic Acids Res, 31, 2751-2758 and Shahbabian, K. et al., The EMBO Journal (2009) 28, 3523-3533). There were also attempts to change the specificity of Ribonuclease T1 and Ribonuclease MC1 (Hoschler, K. et al. J Mol Biol, (1999) 294, 1231-1238, Numata, T. et al., Biochemistry, (2003) 42, 5270-5278). In these two cases the enzyme variants were created in which their specificity has increased, from one to two nucleotides (Numata, T. et al., Biochemistry, (2003) 42, 5270-5278, Czaja, R. et al., Biochemistry, (2004) 43, 2854-2862; Struhalla, M. et al. Chembiochem, (2004) 5, 200-205). However, all these Ribonucleases still have a very limited sequence specificity which makes them unsuitable as molecular biology tools in applications similar to those of DNA restriction enzymes.
Ribonuclease III is an archetype of nucleases that cleave double-stranded RNA (dsRNA) and a founding member of the Ribonuclease III superfamily of proteins, which share an evolutionarily conserved catalytic domain. They are divided into four classes based on the occurrence of additional domains. Class 1, i.e., orthodox Ribonuclease III enzymes, have a double-stranded RNA binding domain (dsRBD) and a single Ribonuclease III domain. Class 2 and 3 enzymes are represented by Drosha and Dicer, respectively, which both comprise two Ribonuclease III domains along with a single dsRBD. In addition, enzymes belonging to class 2 possess additional domains, such as a polyproline domain and to class 3 a DExD helicase, DUF283 and PAZ domains. Class 4, called Mini III, includes enzymes that consist solely of the Ribonuclease III domain.
The natural substrate for the Mini III protein from Bacillus subtilis is 23S pre-rRNA, in which the 3′ and 5′ ends of the molecule are removed to yield the mature 23S rRNA. The cleavage site for this enzyme is known, however close to the cleavage site of double-stranded pre-rRNA one fragment of 23S pre-rRNA forms an irregular helix, which was speculated to be necessary for substrate recognition (Redko, Y. et al., Molecular Microbiology, (2008) 68 (5), 1096-1106). In addition, in vitro endoribonucleolytic activity of Mini III was shown to be stimulated by the ribosomal protein L3 bound to the 3′ end of the 23S rRNA. There is indirect evidence that protein L3 enhances the cleavage of the substrate by changing the conformation of the RNA (Redko, Y. et al., Molecular Microbiology, (2009) 71 (5), 1145-1154).
There are no known enzymes for specific and defined dsRNA fragmentation with properties similar to the DNA restriction endonucleases or DNA nickases (JP 54059392-A, May 12, 1979). The dsRNA can be cleaved by endoribonucleases from Ribonuclease III family, but no details of Ribonuclease III-dsRNA interactions are known (Herskovitz, M. A. et al., Molecular Microbiology, (2000) 38 (5), 1027-1033). The criteria for site-specific binding and selective processing remain unclear (Dasgupta S. et al., Molecular Microbiology, (1998) 28 (3), 629-640). However, unspecific dsRNA endonucleases are used for generating short double-stranded RNA fragments (US 2006057590-A1, Mar. 16, 2006, NOVARTIS). Obtaining enzymes exhibiting sequence specificity in dsRNA cleavage will allow to develop all areas of RNA manipulation techniques, but also to develop new research methods, new applications of such enzymes and new technologies derived from them.
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